Stenotrophomonos maltophilia is an emerging opportunistic Gram-negative pathogen that is increasingly isolated from medical devices and clinical specimens. As one of the top 11 organisms isolated from intensive care unit (ICU) patients, the pathogen poses significant threat to immunocompromised individuals, and is commonly isolated from Cystic Fibrosis (CF) patients. In the debilitated host, S. maltophilia infection most commonly manifests as pneumonia, but the pathogen can also cause bacteremia, endocarditis, urinary tract infection, and skin and soft tissue infections. Treatment o S. maltophilia infection is difficult due to the pathogen's inherent resistance to multiple antibiotics. Currently, our understanding of the virulence mechanisms employed by S. maltophilia to cause disease is very limited. In murine models of pneumonia, S. maltophilia has been shown to grow and persist, elicit an inflammatory response, and cause tissue destruction in the lung. However, the virulence factors that mediate these effects remain to be determined. Our lab has recently identified a type II secretion system in the S. maltophilia strain K279a that mediates cell rounding, actin rearrangement, detachment, and ultimately cell death in the human lung epithelial cell line A549. Preliminary data generated using mutagenesis indicate that the type II secreted serine proteases StmPr1 and StmPr2, which share 40% amino acid sequence identity, contribute to the observed rounding and cell death in A549 cells. Additionally, StmPr1 and StmPr2 are entirely responsible for the serine protease activity observed in cell-free supernatants collected from strain K279a. Both the stmPr1 and stmPr2 loci are found in in the majority of S. maltophilia isolates from CF patients, bolstering the potential clinical relevance o these proteases. Using biochemical and genetic approaches, this proposal aims to i) determine whether or not StmPr1 and StmPr2 are directly involved and sufficient to cause cell rounding; and determine if it is the serine protease activity of StmPr1 and StmPr2 that is responsible for A549 cell rounding, ii) elucidate the mechanism of protease-mediated A549 cell rounding by identifying host targets, and iii) investigate how cell rounding contributes to S. maltophilia pathogenesis by investigating protease-mediated cytotoxic effects and pro-inflammatory responses in a variety of lung cells. Together these aims will further our understanding of protease-mediated disease mechanisms, and will be informative for the development of novel treatment strategies centered on S. maltophilia proteases.